Osteoarthritis (OA) is a major cause of disability in the USA, particularly so in veterans, who are disproportionately affected by aging and joint trauma. OA culminates in failure of the joint, which includes compromised cartilage chondrocyte differentiation and function, by mechanisms that are not completely understood. Because there is no disease-modifying medical therapy for OA, there is major unmet need to advance translation. Our long-term objective is to validate novel targets to limit OA cartilage failure, including processes that promote molecular innate inflammatory processes (inflamm-aging). A major obstacle in the field is that multiple homeostasis mechanisms are dysfunctional in OA chondrocytes, and we need to sort out which are the earliest, and central to the chondrocyte differentiation changes and viability loss. We have identified decreased mitochondrial mass, function, and biogenesis capacity in aging and OA knee chondrocytes, linked partly to deficiency of TFAM and other mitochondrial transcription factors. Our core hypothesis is that articular chondrocyte mitochondrial dysfunction is an early, pivotal, targetable, and reversible change in OA due to aging and biomechanical injury, and amplified by altered mitochondrial retrograde signaling. This includes decrease in the anti-inflammatory mitochondrial peptide humanin, causing effects on chondrocytes that we posit to be at least partly reversible in vitro using the humanin analog HNGF6A. Effective control of injury- and aging-associated tissue degeneration requires not only biogenesis but also maintenance of healthy mitochondria. In the novel, testable OA pathogenesis model that we hypothesize, chondrocyte mitochondrial damage is perpetuated, in large part, by feed-forward and feedback loops involving compromise in cell surveillance mechanisms that normally assure mitochondrial quality control. We specifically hypothesize the failure of chondrocyte mitophagy, via not only deficiency of the mitophagy ?linchpin? BNIP3a, but also decreased proteasomal degradation of damaged polyubiquitinated outer mitochondrial membrane proteins, such as PINK1 and Parkin, by the ubiquitin proteasome system (UPS), which is essential for mitophagy. Our preliminary studies break substantial new ground by revealing markedly impaired UPS function in OA chondrocytes, including defective 20S proteasome core particle proteolytic activity, and accumulation of chondrocyte K48 polyubiquitinated proteins. We further identified that human knee OA chondrocytes have impaired assembly of the proteasome, a state that induces global outcomes of loss of chondrocytic differentiation, via diminished expression of the chondrocyte master transcription factor Sox9, and decreased matrix anabolic gene expression. For testing our integrative model of early, pivotal OA pathogenesis, innovation is applied by our bringing together of a particularly diverse investigative team, including collaborating experts in mitochondrial biology, autophagy and mitophagy, and in the UPS and profiling of cellular ubiquitylome signatures. We will employ our recently validated model of chondrocyte biomechanical injury, and carry out unique studies of human chondrocyte aging, via age-matched analyses of both normal and OA knee chondrocytes. Moreover, we address the problem of specifically testing the role of mitochondrial damage in OA of aging, by generating a chondrocyte-specific mouse model of TFAM knockout, which will be compared to normal mice in analyses for OA with aging in vivo. Completion of these studies will advance our long term goal of spurring translation in OA, by identifying novel OA chondrocyte biomarkers, and by pinpointing humanin and other novel, rational targets for potential development of medical disease-modifying OA therapies.